LOCUS       Exported                8923 bp ds-DNA     circular SYN 06-JAN-2022
DEFINITION  Fluorescent mitophagy reporter.
ACCESSION   .
VERSION     .
KEYWORDS    pCLBW cox8 EGFP mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8923)
  AUTHORS   Rojansky R, Cha MY, Chan DC
  TITLE     Elimination of paternal mitochondria in mouse embryos occurs through
            autophagic degradation dependent on PARKIN and MUL1.
  JOURNAL   Elife. 2016 Nov 17;5. pii: e17896. doi: 10.7554/eLife.17896.
  PUBMED    27852436
REFERENCE   2  (bases 1 to 8923)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2016-11-17"; volume: "5. pii: e17896. doi"; pages: " 
            10.7554/eLife.17896"
FEATURES             Location/Qualifiers
     source          1..8923
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     intron          175..1191
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1115..1137
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1199..1218
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1279..1303
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1310..1384
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1406..2119
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1406..2119
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1448..1469)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1709..1728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2056..2077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2135..2845
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2280..2298)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2548..2567
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2846..2870)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2915..3503
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2968..2988)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3386..3397)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3506..3522)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3507..3523)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3840..4309
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4439..4456)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4610..5198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4690..4709)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5358..6218)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5981..6000
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     enhancer        6370..6749
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6750..6953
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6904..6924
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    7192..7549
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             7614..8030
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7960..7982
                     /label=pLXSN 5
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7998..8014
                     /label=pBABE 5
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    8040..8414
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     8352..8368
                     /label=pBMN 5
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     enhancer        8515..8818
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8820..8923,1..174)
                     /label=chicken beta-actin promoter